PROJECT 2 SUMMARY Idiopathic pulmonary fibrosis (IPF) is an incurable, fatal disease with increasing incidence and mortality. IPF affects at least 5 million people worldwide, with a prevalence as high as 4.3/10,000, and a median survival of 2 to 5 years from diagnosis There is no known cause, no effective treatments and no cure for IPF: one of the few remaining diseases in which this is the case. It is likely that many promising systemic therapies have failed because of poor delivery and penetration inside the lung tissue where they can be most pharmacologically effective. The main objective of this innovative proposal is to determine the degree to which lung-specific delivery of therapeutic agents for IPF will enhance their efficacy. We have discovered that Aminopeptidase 2 (APP2) is highly concentrated in caveolae at the surface of endothelial cells (EC) quite selectively in lung. Caveolae can rapidly, actively, and specifically pump targeted APP2 antibodies out of the blood and across the EC barrier to concentrate them and their attached cargo in underlying lung tissue. This caveolae-mediated transvascular pumping mechanism may enable an ideal delivery system for lung-targeted therapeutics. Based on this novel strategy, we will create new therapeutics that after intravenous injection rapidly and specifically penetrate lung tissue to reach their intended pathophysiological targets and cells. Here, we will leverage caveolae to pump anti- fibrotic agents specifically into lungs during evolving and established fibrosis. We will determine the ability of this caveolar pumping system to target specific relevant cell populations and study their effects on signaling pathways involved in fibrosis, in particular myofibroblast differentiation. We will compare the efficacy of therapeutics relying on standard systemic delivery versus caveolae pumping in three lung fibrosis models. The Thy-1 protein has broad fibrosis-suppressive effects. Its normal expression on lung fibroblast surfaces is lost during fibrosis. Thy-1 inhibits TGF- activation and myofibroblast differentiation in cell culture. Rapamycin also broadly inhibits myofibroblast differentiation and antifibrotic efficacy, but known systemic toxicity limits its clinical use. Pumping Thy-1 and Rapamycin into lungs would be ideal to test antifibrotic activity in vivo. Our hypothesis is that lung-specific delivery of anti-fibrogenic therapies is feasible and will result in enhancement of their therapeutic efficacy. The following specific aims will be tested: Aim 1: To confirm the feasibility of caveolar pumping for targeted delivery in lung disease by characterizing the pumping of caveolae-targeting antibodies with attached cargo (imaging probes, Thy1 and/or nanoparticles) into lungs at distinct stages of fibrosis; Aim 2: To use caveolar pumping to study pulmonary effects of Thy-1 in vivo and to evaluate its therapeutic utility in relevant, complementary animal models of pulmonary fibrosis at distinct stages of disease; Aim 3: To study the ability of the lung-specific caveolar pumping system to enhance the antifibrotic effects and minimize systemic toxicity of rapamycin in vivo in relevant, complementary animal models of pulmonary fibrosis at distinct stages of disease; and Aim 4: To evaluate APP2 expression in human clinical samples from patients with IPF and to evaluate caveolae function and antibody-based delivery ex vivo in human IPF lungs.